GB2127831A - Intermediates in the preparation of caerulein - Google Patents

Abstract

A process for producing a decapeptide of the formula: H-Pyr-Gln-Asp-Tyr-Thr(Ac)-Gly-Trp- Met-Asp-Phe-NH2 (I), which decapeptide is useful as an intermediate in the production of caerulein, which comprises subjecting a hexapeptide represented by the formula: H-Pyr-Gln-Asp-Tyr-Thr(Ac)-Gly- NHNH2 (II> to an azide forming reaction at the C- terminal and then to reaction with a tetrapeptide represented by the formula: H-Trp-Met-Asp-Phe-NH2 (III) The Hexapeptide (II) and its preparation are also disclosed.

Description

1

GB 2 127 831 A 1

SPECIFICATION

Intermediates in the production of caerulein

The present invention relates to intermediates in the production of caerulein and also specifically to a novel process for producing a decapeptide which is an intermediate in the production of caerulein 5 (which has the following structural formula 5

so3h

I

H-Pry-Gin-Asp-Tyr-Thr-Gly-Trp-Met-Asp-Phe-NH2

and was isolated from an extract of the skin of Hylacaerulea, a kind of frog in Australia, by Anastasi et.

al).

It is known that caerulein has activities analogous to those of the hormones of the digestive 10 organs such as, e.g., gastric and pancreozaimine. Thus, it has been used as an agent for diagnosis of 10 drugs for treating progressive paralytic ileus. Lately it has been found that caerulein is effective in the treatment of some mental diseases, particularly, schizophrenia, manic- or depressive psychosis and senile dementia. Thus, research and development with respect to caerulein has been focused its possibilities as a psychotropic drug.

15 Some processes for producing caerulein have been disclosed, for example, in Japanese Patent 15 Publication No. 44—25079 and in Japanese Unexamined Patent Publications Nos. 54—61170, 54—63072 and 55—100348. The most important step in the production of caerulein is sulfonation of the tyrosine residue which is located at the 7th position from the C-terminal of the unsulfonated decapeptide. In the prior art as mentioned above, this sulfonation reaction is achieved with pyridine/S03 20 in anhydrous dimethylformamide or in an aqueous solvent in a prefixed pH-range (pH 8.5—12.5). 20

A further novel and improved process for the sulfonation using S03, whereby treatment of the reaction product after completion of the reaction is easy, comprises using the copolymer of vinylpyridine with divinylbenzene instead of a pyridine base (Japanese Patent Application No. 57—90927). This invention embraces such a process.

25 The decapeptide which may be prepared in this invention is an important intermediate in 25

producing caerulein because it can readily be converted into caerulein by sulfonation of the tyrosine residue which is the 7th amino acid from the C-terminal and subsequent deacetylation of the 6th amino acid, threonine.

The present invention provides a hexapeptide of the formula:

H-Pyr-Gln-Asp-Tyr-Thr(Ac)-Gly-NHNH2;

30 wherein Ac is acyl. Ac may, for example, be acetyl. 30

The hexapeptide of the invention may be used to prepare a decapeptide (mentioned above) which is invaluable in the synthesis of caerulein. The process for preparing the decapeptide is a highly important part of the invention and comprises subjecting the hexapeptide,

H-Pyr-Gln-Asp-Tyr-Thr(Ac)-Gly-NHNH2 (ID

35 to an azide forming reaction at the C-terminal and then reacting the resulting azide with a tetrapeptide 35 of the formula

H-Trp-Met-Asp-Phe~NH2 (HI)

so as to produce a decapeptide of the formula

H-Pyr-Gln-Asp-Tyr-Thr(Ac)-Gly-Trp-Met-Asp-Phe-NH2 (|)

40 The invention also includes a process for preparing caerulein which comprises sulfonating the 40

tyrosine residue and deacylating the threonine residue of a decapeptide of formula (I) produced as above.

The invention further provides a pharmaceutical or veterinary formulation which comprises caerulein which has been prepared as above formulated for pharmaceutical or veterinary use 45 respectively. The present formulations may be in unit dosage form and/or comprise a conventional 45 pharmaceutical^ acceptable or veterinarily acceptable, respectively, diluent, carrier or excipient, in accordance with the normal practice of the skilled man.

The decapeptide (I) of this invention can be produced by condensation of a hexapeptide of formula (II) with a tetrapeptide of formula (III) according to conventional methods. Thus, for example, the C-50 terminal hydrazide of the hexapeptide (II) may be allowed to react with nitrous acid or derivatives 50

thereof to give the corresponding C-terminal azide compound (IV):

GB 2 127 831 A

H-Pyr-Gln-Asp-Tyr-Thr(Ac)-Gly-N3 (IV)

which may then be allowed to react with the tetrapeptide (III). The formation of azide compound (IV)

from hexapeptide (II) may be carried out under known reaction conditions for azide formation; for example, the hexapeptide (II) may be dissolved in a polar solvent such as, for example, dimethyl-5 formamide or dimethylsulfoxide, which is acidified with hydrochloric acid and to which a nitrite such as, 5 for example, sodium nitrite or isoamyl nitrite may be added at a low temperature (e.g. about —10°C to 10°C). The resulting azide compound (IV) may be subjected to reaction with the tetrapeptide (III)

without isolation from the reaction mixture. In more detail, the reaction mixture containing the azide compound (IV) may be basified by the addition of an organic base such as, for example, triethylamine or 10 tri-n-butylamine, a dimethylformamide or dimethylsulfoxide solution of tetrapeptide (II!) added at a low 10 temperature (e.g. about —50 to —20°C, preferably, —30 to —25°C), and the resulting solution allowed to stand at the same temperature for a few days. The objective product (I) may be isolated as a purified compound by means of conventional isolation techniques such as, for example, evaporation of the solvent, reprecipitation, washing with solvents and/or drying.

15 The starting hexapeptide (II) of this invention may be produced from the C-terminal glycine 15

derivative by a process as follows. (The invention includes such a process, the individual peptides, and also processes comprising the appropriate steps set out below for making the individual intermediate peptides. It should be understood that in making hexapeptide (II) the skilled man can start at any desired intermediate stage of the following process and such shorter processes are embraced by the invention.) 20 A threonine derivative represented by the formula: 20

BOC-Thr(Ac)OH (VI)

or a reactive derivative thereof, may be subjected to reaction with a glycylhydrazine derivative represented by the formula:

H-Gly-NHNH-Z (V)

25 to give a dipeptide represented by the formula: 25

BOC-Thr(Ac)-Gly-NHNH-Z (VII)

which after elimination of the protecting group at the N-terminal may be allowed to react with a tyrosine derivative represented by the formula:

BOC-Try(Bzl)OH (VIII)

30 or a reactive derivative thereof to give a tripeptide represented by the formula: 30

BOC-Tyr(Bzl)-Thr(Ac)-Gly-NHNH-Z (IX)

which after elimination of the protecting group at the N-terminal may be allowed to react with an aspartic acid derivative represented by the formula:

BOC-Asp(OBzl)OH (X)

35 or a reactive derivative thereof to give a tetrapeptide represented by the formula: 35

BOC-Asp(OBzl)-Tyr(Bzl)-Thr(Ac)-Gly-NHNH-Z (XI)

which after elimination of the protecting group at the N-terminal may be allowed to react with a glutamine derivative represented by the formula:

BOC-Gln-OH (XII)

40 or a reactive derivative thereof to give a pentapeptide represented by the formula: 40

BOC-Gln-Asp(OBzl)-Tyr(Bzl)-Thr(Ac)-Gly-NHNH-Z (XIII)

which after elimination of the protecting group at the N-terminal may be allowed to react with a pyroglutamic acid derivative represented by the formula:

Z-Pyr-OH

(XIV)

3

GB 2 127 831 A 3

or a reactive derivative thereof to give a hexapeptide represented by the formula:

Z-Pyr-Gln-Asp(OBzl)-Tyr(Bzl)-Thr(Ac)-Gly-NHNH-Z (XV)

which may then be deprotected to give the objective compound (II). In this process the symbols BOC, Z 5 and Bzl mean t-butyloxycarbonyl, benzyloxycarbonyl and benzyl, respectively. g

The hexapeptide (II) may also be produced as shown below. Thus, a tetrapeptide represented by the formula:

H-Pyr-Gln-Asp-Tyr-NHNH2 (XVI)

may be condensed with a dipeptide represented by the formula:

10 H-Thr(Ac)-Gly-NHNH-Z (XVII) 10

by a known method, followed by deprotection of the hydrazide. The invention includes such a process.

Tetrapeptide (III) is a known compound disclosed in J. Chem. Soc., (c) 555 (1966), which may be used in the form of its acid addition salts, e.g. the trifluoroacetate.

The present invention will now be further described and illustrated by way of the following 15' Preparations and Example. The symbols used in the structural formulae below have the same meanings 15> as given above in connection with formulae (I) to (XVII).

Preparation 1 1) BOC-Gly-NHNH-Z:

To a solution of 25 g (0.143 mol) of t-butyloxycarbonylglycine in 300 ml of tetrahydrofuran are 20 added 16.46 g (1.0 mol eq.) of N-hydroxysuccininimide and 2.95 g (1.0 mol eq.) of 20

dicyclohexylcarbodiimide (DCC) with ice-cooling and the mixture is allowed to stand overnight. Then, the precipitated dicyclohexylurea is filtered off and the filtrate is condensed under reduced pressure and crystallized from ethyl acetate/petroleum ether to give 33.09 g (in 85.0% yield) of t-butyloxycarbonylglycine. N-succinimide ester, mp. 160—162°C.

25 The N-succinimide ester is dissolved in a mixture of dimethylformamide (200 ml) and methylene 25

chloride (200 ml) and a solution of 30.58 g (0.184 mol) of benzyloxycarbonylhidradine in 50 ml of methylene chloride is added thereto with stirring and ice-cooling, and the mixture is allowed to stand overnight with ice-cooling. The reaction mixture is condensed under reduced pressure and the residue is dissolved in ethyl acetate, then washed with chilled diluted hydrochloric acid, water, a chilled aqueous 30 solution of sodium bicarbonate and water, successively, and condensed under reduced pressure; the 30 residue is recrystallized from ether petroleum ether to give 56.95 g (in 95.71 % yield) of N-(t-butyloxycarbonylglycyl)-N'-benzyloxycarbonylhydrazine as pillars, m.p. 85—87°C.

Anal. Calcd. (%) for C15H21N305:

C, 55.72; H, 6.55; N, 13.00

35 Found (%): C, 55.61; H, 6.50; N, 13.30 35

2) BOC-Thr(Ac)-Gly-NHNH-Z (VII):

To a solution of 56.95 g (0.176 mol) of the above product in 300 ml of acetic acid is added 300 ml (3 mol eq.) of 1.76 N-dry hydrochloric acid/acetic acid, and after one hour at a room temperature, the mixture is condensed under reduced pressure. To the residue is added 300 ml of toluene, from which 40 most of the acetic acid is removed by azeotropic distillation. Then, 500 ml of ether is added to the 40

residue and the suspension is stirred to give 43.3 g (in 94.75% yield) of N-glycyl-N'-benzyloxycarbonylhydrazine hydrochloride as crystals, m.p. 177—180°C.

Anal. Calcd. (%) for C10H13N303 • HCI:

C, 46.25; H, 5.43; N, 16.18; CI, 13.65

45 Found (%): C, 46.12; H, 5.34; N, 1 5.98; CI, 13.61 45

In 358 ml of dimethylformamide is dissolved 35.84 g (0.138 mol) of the above crystals, to which 329 ml (0.1 mol eq.) of tri-n-butylamine and 49.5 g (0.138 mol) of t-butyloxycarbonyl-O-acetylthreonine • N-succinimide ester [Experientia, 23, 700 (1967)] are added with ice-cooling, and the mixture is allowed to stand at the same temperature for 23 hours. The residue dissolved in ethyl acetate 50 is washed with chilled diluted hydrochloric acid, water, a chilled aqueous solution of sodium 50

bicarbonate and water successively, condensed under reduced pressure and then crystallized from 300 ml of petroleum ether to give 60.25 g (in 93.58% yield) of N-(t-butyloxycarbonyl-0-acetyl-L-

4

GB 2 127 831 A 4

threonylglycyl)-N'-benzyloxycarbonylhydrazine (VII) as crystals, m.p. 54—64°C, M22 +17.0+0.6° (c, 1.01, methanol).

Anal. Calcd. (%) for C21H30N408:

C, 54.07; H, 6.48; N, 12.01

5 Found (%): C, 54.55; H, 6.80; N, 11.52 5

3) BOC-Tyr(Bzl)-Thr(Ac)-Gly-NHNH-Z (IX):

The above product (VII) (32.42 g) [0.0695 mol] is subjected to elimination of the protecting group under the same conditions as in the aforementioned steps to give 26.99g (in 96.39% yield) of N-(0-acetyl-L-threonyl-glycyl)-N'-benzyloxycarbonylhydrazine hydrochloride as crystals, m.p. 64—100°C. 10 [dg2+6.0±0.5° (c, 1.008, methanol) 10

Anal. Calcd. (%) for C16H22N406.HCI:

C, 47.71; H, 5.76; N, 13.91; CI, 8.80

Found (%): C, 47.81; H, 6.28; N, 13.09;. CI, 8.99

To a solution of 26.74 g (0.0664 mol) of the above product in 270 ml of methylformamide are 15 added 12.3 g (1.0 mol eq.) of tri-n-butylamine and 31.1 g (1.0 mol eq.) of t-butyloxycarbonyl-O-benzyl- 15 L-tyrosine • N-succinimide ester with stirring and ice-cooling, and the mixture is allowed to stand overnight. The reaction mixture is condensed under reduced pressure and the residue is dissolved in ethyl acetate, then washed with a chilled diluted hydrochloric acid, water, a chilled aqueous solution of sodium bicarbonate and water, successively, and condensed under reduced pressure. The residue is 20 crystallized from 350 ml of ether to give 31.8 g (in 66.5% yield) of N-(t-butyloxycarbonyl-0-benzyl-L- 20 tyrosyl-0-acetyl-L-threonyl-glycyl)-N'-benzyloxycarbonylhydrazine (IX) as crystals, m.p. 1 67—169°C. [d|2+13.6±0.5° (c, 1.019, methanol)

Anal. Calcd. (%) for C37H,BN5010:

C, 61.74; H, 6.30; N, 9.73

25 Found (%): C, 60.99; H, 6.22; N, 9.54 25

4) BOC-Asp(OBzl)-Tyr(Bzl)-Thr(Ac)-Gly-NHNH-Z (XI):

Under the same conditions as in the above steps, 21.05 g (29.2 mmol) of the above product (IX) is subjected to elimination of the protecting group to give 18.74 g (in 97.6% yield) of N-(0-benzyl-L-tyrosyl-0-acetyl-L-threonyl-glycyi)-N'-benzyloxycarbonylhydrazine hydrochloride, m.p. 120—128°C 30 [a:]22 +21.3±0.6° (c, 1.0, methanol) 30

Anal. Calcd. (%) for C32H37NsOs ■ HCI ■ 1/2H20:

C, 57.78; H, 5.91; N, 10.54; CI, 5.33; H20, 1.35

Found (%): C, 57.63; H, 5.86; N, 10.54; CI, 5.13; H20, 0.69

To a solution of 18.58 g (28.3 mmol) of the above product in 186 ml of dimethylformamide are 35 added 6.7 ml (28.3 mmol) of tri-n-butylamine and 11.9 g (28.3 mmol) of N-succinimide t- 35

butyloxycarbonyl-/3-benzyl-L-aspartate, and the mixture is allowed to stand overnight. The reaction mixture is condensed under reduced pressure, and the residue is solidified with a mixture of ethyl acetate and ether. The resultant solid is collected by filtration, washed with a chilled diluted hydrochloric acid, water, a chilled aqueous solution of sodium bicarbonate and water, successively, then suspended 40 in ethyl acetate, and subjected to filtration to give 24.19g (in 92.0% yield) of N-(t-butyloxycarbonyl-^- 40 benzyl-L-aspartyl-0-benzyl-L-tyrosyl-0-acetyl-L-threonyl-glycyl)-N'-benzyloxycarbonyl hydrazine (XI). m.p. 157—165°C. [a]*2 +1.6±0.4° (c, 1.001, methanol)

Anal. Calcd. (%) for C48HS6N6013:

C, 62.33; H, 6.10; N, 9.09

45 Found (%): C, 61.59; H, 5.97; N, 9.16 45

5) BOC-Gln-Asp(OBzl)-Tyr(Bzl)-Thr(Ac)-Giy-NHNH-Z (XIII)

Under the same conditions as mentioned in the above steps, 24.08 g (25.92 mmol) of the above product (XI) is subjected to elimination of the protecting group to give 22.3 g (in 100% yield) of N-(/J-benzyl-L-aspartyl-O-benzyl-L-tyrosyl-O-acetyl-L-threonyl-glycyD-N'-benzyloxycarbonylhydrazine 50 hydrochloride, [or]22 +19.0±0.6° (c, 1.007, methanol) 50

5

GB 2 127 831 A 5

Anal. Calcd. (%) for C^H^O,, HCI H20:

C, 58.73; H, 5.85; N, 9.56; CI, 4.03; H20, 2.05

Found (%): C, 58.65; H, 5.77; N, 9.78; CI, 3.99; H20, 1.90

To a solution of 22.13 g (25.7 mmol) of the above product in 220 ml of dimethylformamide are 5 added 6.1 ml (1.0 mol eq.) of tri-n-butylamine and 8.82 g (25.7 mmol) of N-succinimide t-

butyloxycarbonyl-L-glutamate under ice-cooling and the mixture is allowed to stand overnight. The reaction mixture is condensed under reduced pressure, and ethyl acetate is added to the residue and stirred. The precipitated solid is collected by filtration, then washed with a chilled diluted hydrochloric acid, water, a chilled aqueous solution of sodium bicarbonate and water, successively, suspended in 10 ethyl acetate and subjected to filtration to give 23.85 g (in 88.0% yield) of N-(t-butyloxycarbonyl-L-glutaminyl-/5-benzyl-L-aspartyl-0-benzyl-L-tyrosyl-0-acetyl-L-threonyl-glycyi)-N'-benzyloxycarbonylhydrazine (XIII). m.p. 219—220°C. [a]£2 —2.1+0.4° (c, 1.007, dimethylformamide)

Anal. Calcd. (%) forCS3H64N8015 1/2 H20:

C, 59.93; H, 6.17; N, 10.55; HzO, 0.85

15 Found {%)■. C, 59.87; H, 6.10; N, 10.87; H20,0.75

6) Z-Pyr-Gln-Asp(OBzl)-Tyr(Bzl)-Thr(Ac)-Gly-NHNH-Z (XV):

Under the same conditions as in the above steps, 23.69 g (22.49 mmol) of the above product (XIII) is subjected to elimination of the protecting group to give 22.41 g (in 100% yield) of N-(L-glutaminyl-/5-benzyl-L-aspartyl-0-benzyl-L-tyrosyl-0-acetyl-L-threonyl-glycyl)-N'-20 benzyloxycarbonylhydrazine hydrochloride. [a]£3 +4.4+0.4° (c, 1.010, dimethylformamide)

Anal. Calcd. (%) for C48H58N8013 HCI H20:

C, 57.22; H, 5.90; N, 11.12; CI, 3.62; H20, 1.79

Found (%): C, 56.85; H, 5.98; N, 11.00; CI, 3.48; H20, 1.19

To a solution of 22.29 g (22.49 mmol) of the above product in 220 ml of dimethylformamide are 25 added 5.4 ml (22.49 mmol) of tri-n-butylamine and 8.1 g (22.49 mmol) of N-succinimide benzyloxycarbonyl-pyroglutamate and the mixture is allowed to stand overnight with ice-cooling. The reaction mixture is condensed under reduced pressure, and 170 ml of ethyl acetate is added to the residue. The precipitated solid is collected by filtration, then washed a chilled diluted hydrochloric acid, water, a chilled aqueous solution of sodium bicarbonate and water, successively, suspended in ethyl 30 acetate and subjected to filtration to give 24.57 g (in 91.3% yield) of N(benzyloxycarbonyl-L-

pyroglutamyl-L-glutaminyl-/3-benzyl-L-aspartyl-0-benzyl-L-tyrosyl-0-acetyl-L-threonyl-glycyi)-N'-benzyloxycarbonylhydrazine (XV). [a;]^3—7.0+0.5° (c, 1.008, dimethylformamide)

Anal. Calcd. (%) for C61H67N9017 ■ 1/2 H20:

C, 60.69; H, 5.68; N, 10.44; H20, 0.74

35 Found (%): C, 59.97; H, 5.84; N, 11.00; H20, 0.76

7) H-Pyr-Gln-Asp-Tyr-Thr(Ac)-Gly-NHNH2 HCI as the hydrochloride of the compound (II):

To a solution of 24.0g (20 mmol) of the above product (XV) in 360 ml of dimethylformamide are added 22.7 ml (2 mol eq.) of 1.76N-dry hydrochloric acid/acetic acid and 53.2 ml of palladium black, and then the mixture is stirred under a hydrogen atmosphere for 6 hours. The palladium catalyst is 40 removed by filtration and washed with dimethylformamide; the filtrate and washings are combined and evaporated under reduced pressure. The residue is solidified from a mixture of methanol and ether to give 16.9 g (in 100% yield) of L-pyroglutamyl-L-glutaminyl-L-aspartyl-L-tyrosyl-O-acetyl-L-threonyl-glycylhydrazide hydrochloride, [a]"-5 —7.2±0.5° (c, 1.016, dimethylformamide)

Anal. Calcd. (%) for C31H43N9013 ■ HCI • H20:

45 C, 46.30; H, 5.77; N, 15.68; CI, 4.53 H20, 2.24

Found (%): C, 45.58; H, 6.30; N, 15.50; CI, 4.51 H20, 2.26

Preparation 2

1) H-Pyr-Gln-Asp-Tyr-Thr(Ac)-Gly-NHNH-Z:

To a solution of 2.69 g of L-pyroglutamyl-L-glutaminyl-L-aspartyl-L-tyrosylhydrazide in a mixture 50 of 7 ml of dimethylsulfoxide and 21 ml of dimethylformamide are added 3.81 ml of 4.88 M hydrochloric acid/acetic acid and 0.55 ml of isoamyl nitrite, and the mixture is allowed to react for 20 minutes at a

5

10

15

20

25

30

35

40

45

50

GB 2 127 831 A

temperature of —23°C 30°C. Then, to this reaction mixture are added a solution of 1.0 g of N-(L-

threonyl-glycy!)-N'-benzyloxycarbonylhydrazine in 6 ml of dimethylformamide and 0.3 ml of tri-n-butylamine, and then the mixture is allowed to react at 4°C for 16 hours. The reaction mixture is worked up in the same manner as in Preparation 1 to give 0.92 g (in 42.2% yield) of N-(L-pyroglutamyl-L-5 giutaminyl-L-aspartyl-L-tyrosyl-O-acetyl-L-threonyl-glycyD-N'-benzyloxycarbonylhydrazine. m.p. 207—208°C (decomposition). [ar]J3 —5.0±0.5° (c, 1.0, dimethylformamide).

Anal. Calcd. (%) for C39H49N9015 • 1/2 H20:

C, 52.46; H, 5.64; N, 14.12; H20, 1.01

Found (%): C, 51.46; H, 5.66; N, 14.54; H20,1.12

10 2) H-Pyr-Gln-Asp-Tyr-Thr(Ac)-Gly-NHNH2 ■ HCI as the hydrochloride of (II): 10

To a solution of 0.781 g of the above product in 15.6 ml of dimethylformamide are added 1 ml of

I.76 N dry hydrochloric acid/acetic acid and 1 ml of palladium black, and the mixture is stirred under a hydrogen atmosphere and then worked up in the same manner as in Preparation 1 to give 0.666 g (in 95.97% yield) of the titled product, hexapeptide hydrazide hydrochloride [the hydrochloride of (II)].

15 EXAMPLE 15

H-Py r-G I n-Asp-Tyr-Th r( Ac)-G ly-T rp-Met-Asp-P he-N H2 (i):

To a solution of 1 5.6 g (16.57 mmol x 1.2) of the above product [the hydrochloride of (II)] in 312 ml of dimethylformamide, which is cooled to —35°C (bath temperature), are added 12.22 ml (1.2 x 3 mol eq.) of dry hydrochloric acid/dioxane (4.9 mol/l) and 3.19 ml (1.2 x 1.2 mol eq.) of isoamyl nitrite, 20 and the mixture is stirred for 20 minutes. The reaction mixture is then cooled to about —45°C, and 20

22.89 ml (1.2 x 4 + 1 mol eq.) of tri-n-butylamine is added dropwise thereto; and then a solution of

II.93 g (19.57 mmol) of L-tryptophyl-L-methyonyl-L-Sspartyl-L-phenylalaninamide trifluoroacetate in 72 ml of dimethylformamide is added thereto at the same temperature, and the mixture is allowed to stand at a temperature of —30°C 25°Cfor 3 days, during which time tri-n-butylamine (11.43 ml as

25 a total amount) is occasionally added dropwise in order to keep the reaction mixture at pH 6—7. The 25 reaction mixture is neutralized with 3.6 ml of acetic acid and condensed under reduced pressure. The residue is suspended in 480 ml of 0.3 N-acetic acid, and the precipitating solid is collected by filtration, washed with water, methanol and ether, successively, and then dried on phosphorus pentoxide under reduced pressure at room temperature for 20 hours to give 17.03 g (in 78% yield) of the objective 30 product L-pyroglutamyl-L-glutaminyl-L-aspartyl-L-tyrosyl-O-acetyl-L-threonyl-glycyl-L-tryptophyl-L- 30 methionyl-L-aspartyl-L-phenyl-alaninamide (I), [a]"— 20.4±1.2° (c,0.508,dimethylformamide)

Anal. Calcd. (%) for C60H75N13019S ■ 2H20:

C, 53.37; H, 5.80; N, 13.48; S, 2.37; H20, 2.67

Found (%): C, 52.68; H, 5.93; N, 13.27; S, 2.42; H20, 3.34

Claims (21)

35 CLAIMS 35

1. A hexapeptide of the formula:

H-Pyr-Gln-Asp-Tyr-Thr(Ac)-Gly-NHNH2;

wherein Ac is acyl.

2. A hexapeptide as claimed in claim 1, wherein Ac is acetyl.

40

3. A process for preparing a hexapeptide as claimed in claim 1, which process comprises either; (a) 40 reacting a threonine derivative of the formula

B0C-Thr(Ac)0H,

or a reactive derivative thereof, with a glycylhydrazine derivative of the formula

H-Gly-NHNH-Z,

45 to give a dipeptide of the formula

BOC-Th r( Ac)-G ly-N H N H-Z,

eliminating from the said dipeptide the protecting group at the N-terminal and reacting the thus-deprotected dipeptide with a tyrosine derivative of the formula

B0C-Try(Bzl)0H,

7

GB 2 127 831 A 7

or a reactive derivative thereof, to give a tripeptide of the formula

BOC-Tyr(Bzl)-Thr(Ac)-Gly-NHNH-Z,

eliminating from the said tripeptide the protecting group at the N-terminal and reacting the thus-deprotected tripeptide with an aspartic acid derivative of the formula

5 BOC-Asp(QBzl)OH, 5

or a reactive derivative thereof, to give a tetrapeptide of the formula

BOC~Asp(OBzl)-Tyr(Bzl)-Thr(Ac)-Gly-NHNH-Z,

eliminating from the said tetrapeptide the protecting group at the N-terminal and reacting the thus-. deprotected tetrapeptide with a glutamine derivative of the formula

10 BOC-Gln-OH, 10

or a reactive derivative thereof, to give a pentapeptide of the formula

BOC-Gln-Asp(OBzl)-Tyr(Bzl)-Thr(Ac)-Gly-NHNH-Z,

eliminating from the said pentapeptide the protecting group at the N-terminal and reacting the thus-deprotected pentapeptide with a pyroglutamic acid derivative of the formula

15 Z-Pyr-OH, 15

or a reactive derivative thereof, to give a hexapeptide of the formula

Z-Pyr-Gln-Asp(OBzl)-Tyr(Bzl)-Thr(Ac)-Gly-NHNH-Z,

and deprotecting the said hexapeptide, wherein BOC, Z and Bzl mean t-butyloxycarbonyl,

benzyloxycarbonyl and benzyl, respectively; or (b) condensing a tetrapeptide of the formula

20 H-Pyr-Gln-Asp-Tyr-NHNH2, 20

or acid addition salt thereof, with a dipeptide of the formula

H-Th r( Ac)-G ly-N H N H -Z,

and deprotecting the resulting protected hydrazide; or in the case of (a) above commencing the process at any intermediate stage and continuing as defined above.

25

4. A process as claimed in claim 3 and substantially as hereinbefore described in either of the 25 foregoing Preparations.

5. A process for producing a decapeptide suitable for use in the preparation of caerulein, which process comprises subjecting a hexapeptide as claimed in claim 1 or claim 2 or which has been prepared by a process as claimed in claim 3 or claim 4 to an azide forming reaction at the C-terminal

30 and then reacting the resulting azide with a tetrapeptide of the formula 30

H-Trp-Met-Asp-Phe-NH2 so as to produce a decapeptide of the formula

H-Pyr-Gln-Asp-Tyr-Thr(Ac)-Gly-Trp-Met-Asp-Phe-NH2

6. A process as claimed in claim 5, wherein the azide forming reaction comprises reacting the C-

35 terminal hydrazide of the hexapeptide with nitrous acid or a derivative thereof. 35

7. A process as claimed in claim 5 or claim 6, wherein the hexapeptide azide is not isolated from the reaction mixture of the azide forming reaction prior to reaction with the tetrapeptide.

8. A process as claimed in claim 5 and substantially as hereinbefore described.

9. A process as claimed in claim 5 and substantially as hereinbefore described in the foregoing

40 Example. 40

10. A process for preparing caerulein which comprises sulfonating the tyrosine residue and deacylating the threonine residue of a decapeptide which has been produced by a process as claimed in any one of claims 5 to 9.

8

GB 2 127 831 A 8

11. A process as claimed in claim 10, wherein the sulfonation is achieved with pyridine/S03 in anhydrous dimethylformamide or in an aqueous solvent in a prefixed pH range of 8.5 to 12.5.

12. A process as claimed in claim 10, wherein the sulfonation is achieved with S03 and a copolymer of vinylpyridine with divinylbenzene.

5

13. A pharmaceutical or veterinary formulation which comprises caerulein which has been 5

prepared by a process as claimed in any one of claims 10 to 12 formulated for pharmaceutical or veterinary use, respectively.

14. A formulation as claimed in claim 13 and in unit dosage form.

15. A formulation as claimed in claim 13 or claim 14 also comprising a conventional

10 pharmaceutical^ acceptable or veterinarily acceptable, respectively, diluent, carrier or excipient. 10

16. An azide compound of formula IV given hereinbefore.

17. A process for preparing a compound as claimed in claim 16 and substantially as hereinbefore described.

18. A peptide of any of the formulae VII, IX, XI, XIII or XV given hereinbefore.

15

19. A process for preparing a peptide as claimed in claim 18 which comprises effecting the 15

appropriate step or steps of process (a) as defined in claim 3.

20. A process for preparing a peptide as claimed in claim 18 and substantially as hereinbefore described.

21. A decapeptide of formula I given hereinbefore.

Printed for Her Majesty's Stationery Office by the Courier Press, Leamington Spa. 1984. Published by the Patent Office, 25 Southampton Buildings, London, WC2A 1AY, from which copies may be obtained.